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Peer-reviewed

Isolation of Neutralizing Antibody

中和抗体的分离

Mayo Yasugi Mayo Yasugi
Kazuyoshi Ikuta Kazuyoshi Ikuta

发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.1005 浏览次数: 9798

评审: Anonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Feb 2013

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Abstract

Use of monoclonal antibodies (MAbs) is an established laboratory strategy for characterization of specific pathogens and their antigenicity. Especially, Human MAbs (HuMAbs) with neutralizing activity against specific virus could have potential therapeutic application, and provide significant information on human epitopes that could be important for developing the next generation of universal vaccines against the virus. In addition to the classical method for murine MAb preparation, several methods for the preparation of HuMAbs have been developed. Here, we describe the development of neutralizing HuMAbs against specific virus. HuMAbs are established by fusion of the peripheral blood mononuclear cells of vaccinated volunteers or patients with the fusion partner cell line, named SPYMEG. Then each of prepared HuMAbs is confirmed whether it can neutralize the specific virus by in vitro neutralization assay.

Keywords: Influenza (流感) search Hemagglutinin (血凝素) search Monoclonal antibody (单克隆抗体) search Neutralization (中和) search Vaccine (疫苗) search

Materials and Reagents

  1. Human blood immunized by vaccination or natural infection against specific pathogen
  2. Human fusion partner SPYMEG cells (product of the Medical & Biological Laboratories Corporation, Ltd, Nagoya, Japan)
  3. Madin-Darby canine kidney (MDCK) cells provided from RIKEN cell bank
  4. MDCK-propagated Influenza B viruses (B/Florida/06/2004 and B/Malaysia/2506/2006) by the National Institute for Infectious Diseases, Japan
  5. Phosphate-buffered saline without Ca2+ and Mg2+ (PBS)
  6. HetaSep (STEMCELL Technologies, catalog number: 07906 )
  7. Ficoll-Paque PLUS (GE, catalog number: 17-1440-03 )
  8. Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, catalog number: 11995 )
  9. Polyethylene glycol #1500 (PEG) (Roche Diagnostics, catalog number: 00-783-641-00 )
  10. Fecal Bovine Serum (FBS) (MP Biomedicals, catalog number: 2917054 )
  11. HAT supplement (Life Technologies, catalog number: 21060 )
  12. Antibody against the specific pathogen
  13. HT supplement (Life Technologies, catalog number: 11067 )
  14. Minimal Essential Medium (MEM) (Sigma-Aldrich, catalog number: M4655 )
  15. Ethanol (Nacalai Tesque, catalog number: 14713-53 )
  16. Hybridoma SFM (Life Technologies, catalog number: 12045-084 )
  17. HiTrap Protein G HP (GE, catalog number: 17-0404-01 )
  18. BM condimed (Roche Diagnostics, catalog number: 10-663-573-001 )
  19. Heparin 5,000 units per 5 ml (Novo-Heparin, Mochida Phrrmaceutical)
  20. FITC-conjugated anti-human IgG (Jackson ImmunoResearch Laboratories, catalog number: 109-096-097 )
  21. DMEM-FBS medium (see Recipes)
  22. DMEM-HAT medium (see Recipes)
  23. DMEM-HT medium (see Recipes)
  24. PBS (see Recipes)

Equipment

  1. 75 cm2 cell culture flask (T-75 flask) (IWAKI PUMPS, catalog number: 3110-075 )
  2. 15- and 50-ml plastic tube (BD Biosciences,, catalog number: 352096 and 352070 )
  3. 6- and 10-cm culture dish (IWAKI PUMPS, catalog number: 3010-060 and 3020-100 )
  4. 6-, 12-, 24-, 48- and 96-well cell culture plate (IWAKI PUMPS, catalog number: 3810-006 , 3815-012 , 3820-024 , 3830-048 and 3860-096 )
  5. Light microscope
  6. Fluorescent microscope
  7. CO2 incubator (5% CO2, 37 °C)
  8. Incubator
  9. Peristaltic pump (Perista BioMini Pump; ATTO Corporation)
  10. Slide A Lyzer Dialysis Cassetes (MW = 10 K) (Thermo Fisher Scientific, model: 2160728 )
  11. Beaker
  12. Stirrer
  13. Stirrer bar
  14. Millex-HV Filter Unit 0.45 μm (EMD Millipore, model: SLHVJ13SL )
  15. 2.5-ml syringe
  16. Floater
  17. 21 G needle
  18. Centrifuge (TOMY DIGITAL BIOLOGY, model: LC-121 ; TS-7 rotor )

Procedure

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Yasugi, M. and Ikuta, K. (2013). Isolation of Neutralizing Antibody. Bio-protocol 3(24): e1005. DOI: 10.21769/BioProtoc.1005.
  2. Yasugi, M., Kubota-Koketsu, R., Yamashita, A., Kawashita, N., Du, A., Sasaki, T., Nishimura, M., Misaki, R., Kuhara, M., Boonsathorn, N., Fujiyama, K., Okuno, Y., Nakaya, T. and Ikuta, K. (2013). Human monoclonal antibodies broadly neutralizing against influenza B virus. PLoS Pathog 9(2): e1003150.

    3. ris ris Download Citation in RIS Format

分类

免疫学 > 抗体分析 > 抗体功能

微生物学 > 抗微生物试验 > 杀伤试验

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